Thomas R. Neu

The EPS Matrix: The “House of Biofilm Cells”

In response to a suggestion by the Biofilms 2007 organizing committee to hold an evening session on biofilm extracellular polymeric substances (EPS), an exceptionally inspiring event followed contributions by Ken Bayles, Alan Decho, Martina Hausner, Jan Kreft, Thomas Neu, Per Nielsen, Ute Romling,

What are Bacterial Extracellular Polymeric Substances

The vast majority of microorganisms live and grow in aggregated forms such as biofilms and flocs (“planktonic biofilms”). This mode of existence is lumped in the somewhat inexact but generally accepted expression “biofilm”. The common feature of all these phenomena is that the microorganisms are embedded in a matrix of extracellular polymeric substances (EPS). The production of EPS is a general property of microorganisms in natural environments and has been shown to occur both in prokaryotic (Bacteria, Archaea) and in eukaryotic (algae, fungi) microorganisms. Biofilms containing mixed populations of these organisms are ubiquitously distributed in natural soil and aquatic environments, on tissues of plants, animals and man as well as in technical systems such as filters and other porous materials, reservoirs, plumbing systems, pipelines, ship hulls, heat exchangers, separation membranes, etc. (Costerton et al. 1987; 1995; Flemming and Schaule 1996). Biofilms develop adherent to a solid surface (substratum) at solid-water interfaces, but can also be found at water-oil, water-air and solid-air interfaces. Biofilms are accumulations of microorganisms (prokaryotic and eukaryotic unicellular organisms), EPS, multivalent cations, biogenic and inorganic particles as well as colloidal and dissolved compounds. EPS are mainly responsible for the structural and functional integrity of biofilms and are considered as the key components that determine the physicochemical and biological properties of biofilms.

Assessment of lectin-binding analysis for in situ detection of glycoconjugates in biofilm systems.

An assessment of lectin-binding analysis for the characterization of extracellular glycoconjugates as part of the extracellular polymeric substances in environmental microbial communities was performed using fully hydrated river biofilms. The applicability of the method was evaluated for single, dual and triple staining with a panel of fluor-conjugated lectins. It was shown that lectin-binding analysis was able to stain glycoconjugates within biofilm communities. Lectin staining also demonstrated spatial heterogeneity within the biofilm matrix. Furthermore, the application of two or even three lectins was possible if suitable combinations were selected. The lectin-binding analysis can be combined with general nucleic acid stains to collect both nucleic acid and glycoconjugate signals. The effects of incubation time, lectin concentration, fluor labelling, carbohydrate inhibition, order of addition and lectin interactions were studied. An incubation time of 20 min was found to be sufficient for completion of lectin binding. It was not possible to ascertain saturating concentration for individual lectins, therefore a standard concentration was used for the assay. Carbohydrate inhibition tests indicated that fluorescein isothiocyanate (FITC)-conjugated lectins had more specific binding characteristics than tetramethyl rhodamine isothiocyanate (TRITC)- or cyanine dye (CY5)-labelled lectins. The order of addition and the nature of the fluor conjugate were also found to influence the binding pattern of the lectins. Therefore the selection of a panel of lectins for investigating the EPS matrix must be based on a full evaluation of their behaviour in the biofilm system to be studied. Despite this necessity, lectin-binding analysis represents a valuable tool to examine the glycoconjugate distribution in fully hydrated biofilms. Thereby, chemical heterogeneities within extracellular biofilm locations can be identified in order to examine the role (e.g. sorption properties, microenvironments, cell-extracellular polymeric substance interactions) of the extracellular polymeric substances in environmental biofilm systems.

Selective degradation of ibuprofen and clofibric acid in two model river biofilm systems

A field survey indicated that the Elbe and Saale Rivers were contaminated with both clofibric acid and ibuprofen. In Elbe River water we could detect the metabolite hydroxy-ibuprofen. Analyses of the city of Saskatoon sewage effluent discharged to the South Saskatchewan river detected clofibric acid but neither ibuprofen nor any metabolite. Laboratory studies indicated that the pharmaceutical ibuprofen was readily degraded in a river biofilm reactor. Two metabolites were detected and identified as hydroxy- and carboxy-ibuprofen. Both metabolites were observed to degrade in the biofilm reactors. However, in human metabolism the metabolite carboxy-ibuprofen appears and degrades second whereas the opposite occurs in biofilm systems. In biofilms the pharmacologically inactive stereoisomere of ibuprofen is degraded predominantly. In contrast, clofibric acid was not biologically degraded during the experimental period of 21 days. Similar results were obtained using biofilms developed using waters from either the South Saskatchewan or Elbe River. In a sterile reactor no losses of ibuprofen were observed. These results suggested that abiotic losses and adsorption played only a minimal role in the fate of the pharmaceuticals in the river biofilm reactors.

Phylogenetic Composition, Spatial Structure, and Dynamics of Lotic Bacterial Biofilms Investigated by Fluorescent in Situ Hybridization and Confocal Laser Scanning Microscopy

A bstractThe phylogenetic composition, three-dimensional structure and dynamics of bacterial communities in river biofilms generated in a rotating annular reactor system were studied by fluorescent in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). Biofilms grew on independently removable polycarbonate slides exposed in the reactor system with natural river water as inoculum and sole nutrient and carbon source. The microbial biofilm community developed from attached single cells and distinct microcolonies via a more confluent structure characterized by various filamentous bacteria to a mature biofilm rich in polymeric material with fewer cells on a per-area basis after 56 days. During the different stages of biofilm development, characteristic microcolonies and cell morphotypes could be identified as typical features of the investigated lotic biofilms. In situ analysis using a comprehensive suite of rRNA-targeted probes visualized individual cells within the alpha-, beta-, and gamma-Proteobacteria as well as the Cytophaga–Flavobacterium group as major parts of the attached community. The relative abundance of these major groups was determined by using digital image analysis to measure specific cell numbers as well as specific cell area after in situ probing. Within the lotic biofilm community, 87% of the whole bacterial cell area and 79% of the total cell counts hybridized with a Bacteria specific probe. During initial biofilm development, beta-Proteobacteria dominated the bacterial population. This was followed by a rapid increase of alpha-Proteobacteria and bacteria affiliated to the Cytophaga–Flavobacterium group. In mature biofilms, alpha-Proteobacteria and Cytophaga–Flavobacteria continued to be the prevalent bacterial groups. Beta-Proteobacteria constituted the morphologically most diverse group within the biofilm communities, and more narrow phylogenetic staining revealed the importance of distinct phylotypes within the beta1-Proteobacteria for the composition of the microbial community. The presence of sulfate-reducing bacteria affiliated to the Desulfovibrionaceae and Desulfobacteriaceae confirmed the range of metabolic potential within the lotic biofilms.

Advanced imaging techniques for assessment of structure, composition and function in biofilm systems

Scientific imaging represents an important and accepted research tool for the analysis and understanding of complex natural systems. Apart from traditional microscopic techniques such as light and electron microscopy, new advanced techniques have been established including laser scanning microscopy (LSM), magnetic resonance imaging (MRI) and scanning transmission X-ray microscopy (STXM). These new techniques allow in situ analysis of the structure, composition, processes and dynamics of microbial communities. The three techniques open up quantitative analytical imaging possibilities that were, until a few years ago, impossible. The microscopic techniques represent powerful tools for examination of mixed environmental microbial communities usually encountered in the form of aggregates and films. As a consequence, LSM, MRI and STXM are being used in order to study complex microbial biofilm systems. This mini review provides a short outline of the more recent applications with the intention to stimulate new research and imaging approaches in microbiology.

Application of multiple parameter imaging for the quantification of algal, bacterial and exopolymer components of microbial biofilms

Abstract Techniques are required for the simultaneous or sequential determination of multiple parameters within microbial biofilms. Confocal scanning laser microscopy in combination with a range of fluorescent probes and markers offers an approach to quantitatively defining many aspects of biofilm communities. By applying multispectral imaging in conjunction with nucleic acid stains, fluor conjugated lectins, and autofluorescence we have developed a simple approach to evaluate biofilm community composition. Biofilms were treated with the fluorescent nucleic acid stain SYTO 9 to allow quantification of bacterial biomass and fluor conjugated lectins (i.e., Triticum vulgaris lectin) to identify and allow quantification of exopolymeric substances. Far red autofluorescence was imaged to quantify algal biomass. Digital image analysis of the CSLM optical thin sections in each of the channels was used to determine such parameters as biofilm depth, bacterial cell area (biomass), exopolymer area and algal biomass at various depths and locations. In addition, three colour red–green–blue projections of the biofilms were computed. The method proved simple and effective for determining treatment effects such as grazing by invertebrates.

Microscale and Molecular Assessment of Impacts of Nickel, Nutrients, and Oxygen Level on Structure and Function of River Biofilm Communities

ABSTRACT Studies were carried out to assess the influence of nutrients, dissolved oxygen (DO) concentration, and nickel (Ni) on river biofilm development, structure, function, and community composition. Biofilms were cultivated in rotating annular reactors with river water at a DO concentration of 0.5 or 7.5 mg liter−1, with or without a combination of carbon, nitrogen, and phosphorus (CNP) and with or without Ni at 0.5 mg liter−1. The effects of Ni were apparent in the elimination of cyanobacterial populations and reduced photosynthetic biomass in the biofilm. Application of lectin-binding analyses indicated changes in exopolymer abundance and a shift in the glycoconjugate makeup of the biofilms, as well as in the response to all treatments. Application of the fluorescent live-dead staining (BacLight Live-Dead staining kit; Molecular Probes, Eugene, Oreg.) indicated an increase in the ratio of live to dead cells under low-oxygen conditions. Nickel treatments had 50 to 75% fewer ‘live’ cells than their corresponding controls. Nickel at 0.5 mg liter−1 corresponding to the industrial release rate concentration for nickel resulted in reductions in carbon utilization spectra relative to control and CNP treatments without nickel. In these cases, the presence of nickel eliminated the positive influence of nutrients on the biofilm. Other culture-dependent analyses (plate counts and most probable number) revealed no significant treatment effect on the biofilm communities. In the presence of CNP and at both DO levels, Ni negatively affected denitrification but had no effect on hexadecane mineralization or sulfate reduction. Analysis of total community DNA indicated abundant eubacterial 16S ribosomal DNA (rDNA), whereas Archaea were not detected. Amplification of the alkB gene indicated a positive effect of CNP and a negative effect of Ni. The nirS gene was not detected in samples treated with Ni at 0.5 mg liter−1, indicating a negative effect on specific populations of bacteria, such as denitrifiers, resulting in a reduction in diversity. Denaturing gradient gel electrophoresis revealed that CNP had a beneficial impact on biofilm bacterial diversity at high DO concentrations, but none at low DO concentrations, and that the negative effect of Ni on diversity was similar at both DO concentrations. Notably, Ni resulted in the appearance of unique bands in 16S rDNA from Ni, DO, and CNP treatments. Sequencing results confirmed that the bands belonged to bacteria originating from freshwater and marine environments or from agricultural soils and industrial effluents. The observations indicate that significant interactions occur between Ni, oxygen, and nutrients and that Ni at 0.5 mg liter−1 may have significant impacts on river microbial community diversity and function.

Effect of flow regime on the architecture of a Pseudomonas fluorescens biofilm.

A comparison of the effects of laminar versus turbulent flow regime on the characteristics of a single-species biofilm is presented. The study was carried out by growing Pseudomonas fluorescens biofilms in a flow cell and studying the different layers of the biological matrix with a confocal laser-scanning microscope. The following conclusions were obtained: i) a higher concentration of cells was found in the upper layers of the microbial films than in their inner layers, regardless of the flow regime; ii) the fraction of cells in the overall biofilm mass decreased with time as the film grew; and iii) under laminar flow the total number of cells was higher than in biofilms formed under turbulent flow, but the latter had a higher number of cells per unit volume. Such conclusions, together with the fact that the biofilms were more dense and stable when formed in contact with turbulent flows, favor the design of more compact and efficient biofilm reactors operating in turbulent conditions.

Confocal laser scanning microscopy for analysis of microbial biofilms.

Publisher Summary Among the most versatile and effective of the nondestructive approaches for studying biofilms is confocal laser scanning microscopy (CLSM). CLSM reduces greatly the need for pretreatments such as disruption and fixation that reduce or eliminate the evidence for microbial relationships, complex structure, and organization in biofilms. CLSM has also been used extensively in combination with fluorescent in situ hybridization techniques. CLSM provides a digital database that is amenable to image processing and analysis. Thus the user may obtain quantitative information on a wide variety of parameters, including cell numbers, cell area, object parameters such as minimum/maximum dimensions, orientation, and average gray value. This chapter aims to provide a primer for CLSM observation of biofilm materials. CLSM offers the only means for real-time, in-depth analysis of undisturbed biofilms.